Type IV P-type ATPases (P4-ATPases) are a large family of putative phospholipid translocases, or flippases, implicated in the generation and maintenance of phospholipid asymmetry in biological membranes. It is thought that P4-ATPases directly pump specific lipid substrates, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the extracellular leaflet to the cytosolic leaflet of a membrane to produce asymmetry. The medical significance of an asymmetric plasma membrane is best understood in blood cells where regulated exposure of phosphatidylserine (PS) on the extracellular leaflet induces blood clotting. In addition, cells undergoing programmed cell death also expose PS on the extracellular leaflet facilitating their recognition and phagocytosis by other cells. Thus, normal establishment and regulation of membrane phospholipid asymmetry plays a critical role in prevention of cardiovascular disease and in tissue remodeling during development and wound repair. Moreover, deficiency of a human P4-ATPase (Atp8b1) causes familial intrahepatic cholestasis, a disease where loss of PS asymmetry in the bile canalicular membrane leads to damage of this membrane by secreted bile, ultimately leading to liver failure. P4-ATPase deficiency in mice is linked to diet induced obesity and type 2 diabetes (Atp10a and Atp10d) as well as decreased male fertility (Atp8b3). Characterization of P4-ATPases in the budding yeast Saccharomyces cerevisiae (Drs2, Neo1, Dnf1, Dnf2 and Dnf3) has allowed the application of powerful molecular genetic tools to dissect the biochemical and cell biological functions of these potential flippases. In addition to supporting the proposed function in generating membrane asymmetry, these studies have surprisingly shown that P4-ATPases are required for vesicle-mediated protein transport in the secretory and endocytic pathways. Drs2 localizes to the trans-Golgi network (TGN) and is required to bud AP-1/clathrin-coated vesicles from this organelle by a mechanism that is independent of clathrin coat recruitment to the membrane. It is hypothesized that Drs2 directly pumps phospholipid substrates across the TGN membrane to induce membrane curvature that is captured and molded by clathrin into a vesicle. The proposed studies will determine for the first time if a P4-ATPase (Drs2) is sufficient in a purified form to directly catalyze phospholipid flippase activity in proteoliposomes. The native substrate preference will be determined in the reconstituted system as well as the contribution of the noncatalytic subunit (Cdc50) to flippase activity. The influence of Drs2 activity on membrane curvature and vesicle formation with proteoliposomes and isolated TGN membranes will be tested. In addition, preliminary studies indicate that Drs2 is a novel effector of important molecules controlling vesicle budding from the TGN (phosphatidylinositol 4-phosphate, ArfGEF, Kes1) and the mechanistic basis for this regulation will be determined. PUBLIC HEALTH RELEVANCE: Deficiencies in type IV P-type ATPases (P4-ATPases) are linked to liver disease, obesity and type II diabetes. The precise biochemical and cell biological function of P4-ATPases is still uncertain, although a growing body of evidence suggests that these pumps are phospholipid flippases that control membrane phospholipid asymmetry and vesicle- mediated protein transport. The proposed studies will determine if a P4-ATPase catalyzes phospholipid flippase activity and will define the molecular mechanisms for how a P4- ATPase activity is coupled to the budding of clathrin-coated vesicles from the Golgi complex.